The long-term goals of this project are to elucidate the role and regulation of RhoA signaling pathways during HIV-1 replication and pathogenesis, and to validate a novel host (p115 RhoGEF) and viral (gp41C) targets for anti-HIV therapies. The RhoA GTPase is involved in regulating multiple cellular processes via multiple effector pathways. We have recently discovered that the cytoplasmic domain of the HIV-1 transmembrane protein gp41 (gp41C) interacts with and inhibits p115-RhoGEF, which is a specific guanine nucleotide exchange factor (GEF) and activator of RhoA. The interaction is genetically correlated with enhanced HIV-1 replication in vitro and in vivo. We have further demonstrated that activation of RhoA by p115-RhoGEF or its specific G protein activator (Galpha13) inhibits HIV-1 replication. The inhibitor activity of RhoA is genetically seperable from its activities in activation of actin stress fiber formation, cellular transformation and Serum Response Factor (SRF). In addition, we showed that RhoA inhibited HIV gene expression. The NFATc/AP2 binding site and sequences in the Negative Regulatory Element (NRE) region of HIV Long Terminal Repeat (LTR) are required for the inhibition. We further showed that RhoA inhibited NFAT-dependent gene expression in activated T cells. I hypothesize that RhoA inhibits HIV-1 replication via a novel effector involving NFAT in T cells. Derivative corollaries of this hypothesis include 1) p115 RhoGEF is a major activator of the RhoA signaling pathway in T cells; 2) HIV-1 gp41C modulates p115-mediated RhoA activation to optimize HIV-1 replication; and 3) Disruption of the interaction between gp41C and p115 RhoGEF will lead to the development of anti-HIV therapeutics. The specific aims are as follows. First, we will define the novel RhoA effector function involved in suppressing HIV-1 replication. In addition to a novel signaling pathway involved in HIV-1 replication, the unique RhoA effector will also help to understand the important role of RhoA signaling in various cell signaling processes. Second, we propose to elucidate the mechanism of RhoA-mediated suppression of HIV-1 gene expression. We will define a novel RhoA-mediated mechanism of NFATc (and AP2?) regulation. Third, we will study the functional interaction between p115 RhoGEF and HIV-1 gp41C. We will define the structure of HIV-1 gp41C and p115 involved in their interaction and its function in HIV replication. HIV mutants with loss of interaction between gp41C and p115 will be studied to elucidate the mechanism of gp41C/p115 interaction in HIV-1 replication in vitro and in vivo. The findings will also shed light on mechanisms of RhoA activation by RhoGEFs, of which very little is known. Finally, the defined gp41C and p115 motifs will serve as novel targets for anti-HIV therapy. The potential therapeutics based on disruption of gp41C/p115 interaction will be analyzed in T cell lines, human fetal thymus organ culture (HF-TOC) and in SCID-hu Thy/Liv mice. The new class of anti-HIV peptides will shed light on the development of therapeutics targeting the HIV-1 gp41C tail. The specific RhoA effector, its regulation by p115 or gp41C, and the downstream transcription factors involved in HIV regulation, will further our understanding of RhoA signaling in HIV replication, T cell activation, apoptosis and development.